The Journal of Immunology
NF-B-Dependent Regulation of the Timing of Activation-Induced Cell Death of T Lymphocytes1 Akanksha Mittal,* Salvatore Papa,‡ Guido Franzoso,‡ and Ranjan Sen2† One of the mechanisms by which activated T cells die is activation-induced cell death (AICD). This pathway requires persistent stimulation via the TCR and engagement of death receptors. We found that TCR stimulation led to transient nuclear accumulation of the NF-B component p65/RelA. In contrast, nuclear c-Rel levels remained high even after extended periods of activation. Loss of nuclear p65/RelA correlated with the onset of AICD, suggesting that p65/RelA target genes may maintain cell viability. Quantitative RNA analyses showed that three of several putative NF-B-dependent antiapoptotic genes were expressed with kinetics that paralleled nuclear expression of p65/RelA. Of these three, ectopic expression only of Gadd45 protected significantly against AICD, whereas IEX-1 and Bcl-xL were much less effective. We propose that the timing of AICD, and thus the length of the effector phase, are regulated by transient expression of a subset of p65/RelA-dependent antiapoptotic genes. The Journal of Immunology, 2006, 176: 2183–2189.
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oordination of T cell proliferation, effector function, and death are crucial in generating effective immune responses (1). Ag recognition by mature T cells triggers a proliferative burst that creates a large pool of effector T cells to combat Ag. This primary expanded population of cells shrinks due to cytokine deprivation and loss of cytokine-dependent antiapoptotic signals (1–3). This form of cell death has been referred to as passive cell death or activated cell autonomous death. Several lines of evidence indicate that Bcl-2 family members regulate the balance between viability and apoptosis at this stage. First, downregulation of Bcl-2 correlates with the onset of apoptosis (4, 5); conversely, transgenic expression of Bcl-2 reduces passive cell death (3, 6 – 8). Second, genetic deletion of the proapoptotic BH3 domain gene, Bim, leads to enhanced survival of these cells (4). The importance of this phenomenon is underscored by widespread systemic inflammation and autoimmunity in Bim-deficient mice (9). The IB-related Bcl3 gene has also been implicated in the survival of T cells activated in the presence of adjuvant (10, 11). A second form of activated T cell death is initiated at cell surface death receptors, such as Fas and TNFR2 (1–3, 6). Exposure of activated T cells to IL-2 results in transcriptional induction of the gene encoding Fas ligand (FasL)3 and its expression on the cell surface (2, 12–14). Reactivation of these cells via the TCR and subsequent Fas/FasL interactions directly activate effector caspases that eventually lead to cell death (2, 15). This form of cell death has been referred to as activation-induced cell death (AICD). *Rosensteil Research Center and Department of Biology, Brandeis University, Waltham, MA 02454; †Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD 21224; and ‡Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637 Received for publication June 29, 2005. Accepted for publication December 8, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
This work was supported in part by National Institutes of Health Grant AI41035 (to R.S.) and the intramural research program of the National Institute on Aging.
2 Address correspondence and reprint requests to Dr. Ranjan Sen, Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD 21224. E-mail address:
[email protected] 3 Abbreviations used in this paper: FasL, Fas ligand; AICD, activation-induced cell death; IAP, inhibitor of apoptosis; NES, nuclear export sequence; PI, propidium iodide; IEX-1, immediate early gene X-1; MSCV, murine stem cell virus.
Copyright © 2006 by The American Association of Immunologists, Inc.
Mutations in Fas or FasL lead to severe lymphoproliferative disorders in humans and mice, indicating the importance of this pathway in guarding against aberrant T cell activation (16 –18). Cell death initiated at Fas plays a minor role in passive cell death and is not rescued by Bcl-2 family proteins (1, 7, 8, 19). Rather, persistent activation via the TCR is believed to be essential to trigger AICD (1, 2, 17). Susceptibility to AICD is influenced by at least two factors. First, freshly activated naive T cells express high levels of FLIP, an inhibitor of Fas-initiated death. Therefore, these cells are not killed by the Fas/FasL pathway. However, FLIP levels decrease with IL-2-induced proliferation, so that repeatedly activated cells express lower levels of FLIP and are therefore more susceptible to Fas-mediated death (13, 14). Second, signaling via the Fas receptor itself is altered in persistently activated cells; unlike naive cells, these cells rapidly activate the death-inducing signaling complex, which leads to activation of caspase 8 (15). It is important to regulate the onset of AICD to maintain a balance between effector phase and eventual cell death. Too short an effector phase could result in inefficient immune responses, whereas too long an effector phase could result in the accumulation of unwanted cells. The mechanism(s) by which this is achieved is not well understood. The inducible transcription factor NF-B (20 –22) has been shown to provide survival signals to lymphocytes via up-regulation of antiapoptotic genes (23, 24). However, the role of NF-B in AICD is complex. A protective role for NF-B during AICD was proposed based on the observation that a dominant negative form of IB␣ that prevents NF-B induction increased AICD in T cell clones (25) and activated T cell blasts (26). This was also observed in transgenic mice that express the mutated IB␣ gene, although in this case developmental irregularities could not be ruled out (25). Additionally, AICD was increased in a mutant Jurkat cell line in which NF-B could not be induced due to a mutation in IKK-␥ (27). In contrast, analysis of c-Rel- or p65/RelAdeficient primary T cells suggests that these Rel family members do not protect against AICD (28 –30). Although involvement of NF-B in regulating life and death is well established, the mechanism(s) by which a balance between NF-B-dependent cell life and eventual cell death is coordinated is relatively unexplored. In this manuscript we provide a model for the regulation of AICD in T lymphocytes. We show that the presence of p65/RelA 0022-1767/06/$02.00
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in the nucleus is transient after TCR stimulation; in contrast, nuclear c-Rel levels remain high even after prolonged times of activation. The timing of AICD coincides closely with the loss of p65 from the nucleus, suggesting that p65-regulated genes may define the period of cell viability. Consistent with this hypothesis, we found that inducible expression of three putative NF-B target genes, Bcl-x, immediate early gene X-1 (IEX-1), and Gadd45, closely matched the kinetics of nuclear p65 expression. Ectopic expression of Gadd45, but not Bcl-xL or IEX-1, significantly prolonged cell viability, suggesting that subcellular dynamics of p65/ RelA may determine the duration of the functional phase of activated T cells before AICD.
The percentage of GFP⫹PI⫺ cells was determined at each time point in both P⫹I/anti-CD3- and anti-CD28-treated and untreated cells. The viability of activated cells was normalized to the viability of unactivated cells at each time point according to the formula: (% treated GFP⫹PI⫺ at t (h))/(% untreated GFP⫹PI⫺ at t (h)) ⫻ 100. The viability of uninfected cells was determined by the percentage of PI⫺ cells in the population. The same formula was used for determining the viability of the uninfected cells.
Extracts and immunoblotting
Materials and Methods
Extracts were prepared as described previously (37, 38). The level of cytoplasmic contamination in the nuclear extracts was assessed by immunoblotting with ␣-tubulin Ab. Abs against p65, c-Rel, IB␣, IB, SP1, and inhibitors of apoptosis-2 (IAP2) were purchased from Santa Cruz Biotechnology. Abs against TFIIB and Bcl-x were obtained from BD Transduction Laboratories, and ␣-tubulin was purchased from MP Biomedicals. Ab to Gadd45 has been previously described (35).
Cells and culture
RNA extraction, RT-PCR, and real-time PCR
DC27 and D5h3 murine CD4ⴙ T cell hybridoma cells and splenic CD4⫹ T cell blasts were maintained in RPMI 1640 supplemented with 10% heatinactivated FBS and antibiotics. BOSC23 were maintained in DMEM with 10% FBS. DC27 was derived by transfecting TCR␣ genes from the T cell clone KB5C20 into D011.10.2 T cell hybridoma (31–33), and D5h3 was derived from Ar-5 cells by fusion with the TCR variant of BW5147 thymoma cells (34). Single-cell spleen suspensions from BALB/c mice, 4 – 8 wk of age, were used to positively select CD4⫹ T cells using anti-CD4 MACS microbeads (Miltenyi Biotec). Cells (2.5 ⫻ 106/ml) were incubated with 4 g/ml Con A (Calbiochem) for 3 days. Then cells were washed extensively to remove Con A and incubated for 3– 4 additional days in IL-2 (10 ng/ml; R&D Systems), after which IL-2 medium was removed, and cells were rested overnight. These cells will be referred to as splenic CD4⫹ T cell blasts. These cells were used for TCR stimulation and AICD assays. The purity of cultured CD4⫹ T cells was 94 –99%, as determined by flow cytometry.
Expression vectors The murine stem cell virus (MSCV) was used to clone the human IEX-1 cDNA (gift from Dr. M. Wu, Boston University School of Medicine, Boston, MA). The IEX-1 fragment was isolated from pcDNA3 using XbaI and BamHI and cloned into XhoI (blunt)/BglII-cut MSCV. The Gadd45 expression vector has been previously described (35). For MIGR1-Bcl-XL, the Bcl-XL fragment was isolated from pcDNA3 using EcoRI (35) and cloned into the MSCV-based vector MIGR1.
AICD induction Splenic CD4⫹ T cell blasts, DC27, and D5h3 cells were treated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (2 M; Calbiochem) or 1 g/ml anti-CD3- and anti-CD28 (BD Pharmingen)-coated plates. Cell aliquots were taken at the indicated time points to measure cell viability, prepare nuclear and cytoplasmic extracts, or prepare RNA.
Retroviral infection Retroviruses were produced by cotransfecting BOSC23 cells with retroviral plasmid DNA and the pCL-Eco packaging plasmid. Cell supernatant was harvested 48 h later. DC27 cells (106) were spin-infected (2500 rpm) with 1 ml of viral supernatant and 4 g/ml polybrene for 1.5 h at 30°C. For DC27 cells, the efficiency of infection was 85–95% GFP⫹ cells. Splenic CD4⫹ T cells were stimulated with Con A for 24 h before being spininfected with 2 ml of viral supernatant as described above. After infection, 80% of the viral supernatant was removed, and cells were incubated in 2 ml of medium containing Con A and IL-2 for 24 h. The complete infection protocol was repeated once more the following day. The day after the second infection, Con A was washed off cells, and they were incubated for 3– 4 days in IL-2. Viable cells were then purified using Lympholyte-M (Cedarlane Laboratories) and rested overnight in fresh medium. The efficiency of infection was assessed using flow cytometry and was found to be ⬃30 – 60% GFP⫹ and ⬎98 –99% CD4⫹.
Cell viability analysis Cells were stained with propidium iodide (PI; Sigma-Aldrich; final concentration, 2.5 g/ml) before FACS. Infected cells were distinguished from uninfected cells by GFP expression. The GFP⫹PI⫺ population represented viable infected T cells (DC27 or splenic CD4⫹ T cell blasts) (36). For each FACS sample, 10,000 –15,000 events were analyzed. Gates were set on the GFP⫹ population, and this gated population was analyzed for PI uptake.
RNA was isolated using ULTRASPEC reagent (Biotecx) after treatment with DNase I amp grade (Invitrogen Life Technologies). cDNA was synthesized using random hexamers and the SuperScript First Strand Synthesis System (Invitrogen Life Technologies). Real-time PCR analysis was performed using a Rotorgene 3000 (Corbett Research). One microliter of cDNA (from a total 20-l reaction) was used in a 10-l real-time PCR. The reactions contained 0.5 U of Platinum Taq DNA polymerase (Invitrogen Life Technologies), 0.1 mM dNTP mixture, 0.25 M of each primer, 1.5 mM MgCl2 and 1⫻ PCR buffer, and 0.2⫻ SYBR Green (Molecular Probes). PCR was performed under the following conditions: 3 min and 30 s at 95°C, followed by 35 cycles of 20 s at 94°C, 30 s at 60°C, and 40 s at 72°C. The primers used in this study were designed using Primer3 software from the Whitehead Institute (具http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi典). The sequences of the primers used to amplify murine genes are as follows: IEX1, 5⬘-TCT GGT CCC GAG ATT TTC AC-3⬘ and 5⬘-ACA CAC CCT CTT CAG CCA TC-3⬘; GAPDH, 5⬘-TGC ACC ACC AAC TGC TTA G-3⬘ and 5⬘-GAT GCA GGG ATG ATG TTC-3⬘; and IAP2, 5⬘-CTC CAA CCT GTG CTC TAG CC-3⬘ and 5⬘-GTC TGC GGT GCT CTG ACA TA-3⬘. Primers to Gadd45 were described by Lu et al. (39), Bcl2 was reported by Tomayko and Cancro (40) and Grossmann et al. (41), and Bcl-x was described by Grillot et al. (42) and Grossmann et al. (41). Samples from each time point were analyzed in triplicate in a single real-time PCR assay using the test gene and the normalizing control gene, GAPDH. The values were averaged to a single value for each sample. The value for the test gene for each time point was normalized to the GAPDH value from the same time point. The normalized values from each time point were then compared with the normalized value at 0 h to obtain the fold induction over the value at 0 h.
Results NF-B dynamics in activated T cells T cell stimulation via Ag receptors induces several transcription factors that mediate effector functions of activated T cells. These include members of the NF-B, NF-AT, and AP-1 families (43). For cells that have been triggered several times, the activated phase is terminated by apoptosis initiated by engagement of cell surface death receptors, such as Fas and TNFR (1, 2). To investigate whether NF-B proteins were involved in the regulation of AICD, we first determined the time course of NF-B activation in T cells. Activation of DC27 T hybridoma cells with PMA and the calcium ionophore, ionomycin, rapidly induced nuclear p65/RelA, which reached a peak at 4 – 6 h and then subsided after an 8-h stimulation (Fig. 1A). Analysis of the same extracts showed that c-Rel levels remained high even after p65/RelA had decreased. The slower kinetics of c-Rel activation relative to p65 are probably the result of p65 being induced by a post-translational mechanism, whereas c-Rel induction requires de novo gene transcription and new protein synthesis (44). Loss of p65 from the nucleus required protein synthesis (data not shown), suggesting that p65/RelA was exported out of the nucleus by newly synthesized IB␣. The effect was most pronounced for p65 (compared with c-Rel), probably because IB␣ and p65 both contain efficient nuclear export sequences
The Journal of Immunology
2185 NF-B decline coincides with onset of cell death AICD in a T cell hybridoma and splenic CD4⫹ T cell blasts was examined by PI exclusion using flow cytometry. Although DC27 T hybridoma cells began to die soon after treatment with PMA and ionomycin or after anti-CD3 plus anti-CD28 cross-linking, the sharpest decline in viability occurred after 10 h of activation (Fig. 2A). This time point coincided closely with minimal nuclear p65 expression during the course of the experiment. To confirm that cell death under these conditions was mediated by the Fas/FasL pathway, we used a Fas-deficient hybridoma, 5D5 (47). These cells did not die in response to either PMA and ionomycin or anti-CD3 and anti-CD28 cross-linking (Fig. 2A). In splenic CD4⫹ T cell blasts, a low level of cell death was evident even in unstimulated
FIGURE 1. Transient p65/RelA induction in activated T cells. DC27 T hybridoma cells (A) or D5h3 T hybridoma cells (B) were treated with PMA and ionomycin (P⫹I) for the times indicated above each panel. Nuclear extracts were fractionated by SDS-PAGE and transferred to membranes, which were probed with Abs to p65/RelA, c-Rel, and SP1 or TFIIB, as indicated. Splenic CD4⫹ T cell blasts were activated with P⫹I (C) or Abs to CD3 and CD28 (D) for the times indicated. Equal amounts of nuclear extracts were fractionated by SDS-PAGE and immunoblotted using the indicated Abs. SP1 and TFIIB were used as loading controls for nuclear extracts. The data shown are representative of two or three independent experiments.
(NES) (37, 38, 45, 46). Not only is there no NES in c-Rel that increases its nuclear propensity compared with p65, but it is also likely that new c-Rel synthesis exceeds the amount of newly synthesized IB␣, which results in its predominant nuclear localization at a time when nuclear p65 levels have been considerably reduced. The pattern of transient p65 and prolonged c-Rel induction was confirmed in a second CD4⫹ T cell hybridoma cell line D5h3 (Fig. 1B). Transient nuclear p65 expression was also evident in untransformed CD4⫹ T cell blasts. To restrict the analysis to secondary stimulation, we used spleen CD4⫹ T cell blasts expanded in vitro for 3 days in IL-2. The cells were rested for 12–16 h before activation with PMA and ionomycin. Analysis of nuclear extracts by Western blots showed transient p65/RelA induction, although the kinetics differed somewhat from those in hybridoma cell lines (Fig. 1C). During the same time course, the c-Rel level fluctuated much less, although we reproducibly observed a slight decrease at ⬃8 h when the p65 level was close to that in unstimulated cells. This time point coincided closely with maximum re-expression of IB␣ after signal-induced degradation (data not shown). IB levels were affected much less during the time course of the experiment. In CD4⫹ T cell blasts activated with anti-CD3 plus anti-CD28 cross-linking, p65 levels were maximal after 2– 6 h of activation, whereas c-Rel levels remained high even after 10 h (Fig. 1D). We conclude that activation via the TCR leads to transient nuclear accumulation of p65/RelA and stable accumulation of c-Rel.
FIGURE 2. AICD coincides with a decrease in nuclear p65/RelA. DC27 T hybridoma cells (A) or splenic CD4⫹ T cell blasts were treated with PMA and ionomycin (B) or with CD3 and CD28 Abs (C) or were left untreated for the indicated times (x-axis) and assayed for viability using PI and flow cytometry as described in Materials and Methods. The percent viability (y-axis) indicates the proportion of PI-negative cells in the population. Results represent the mean ⫾ SD from at least three independent experiments; each experiment was performed in triplicate for every sample and condition.
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cells over the time course of the experiment. Cells activated with PMA and ionomycin showed significantly reduced viability after 4 h of activation (Fig. 2B). Those treated with anti-CD3 and antiCD28 showed a sharp decline in viability after ⬃8 h (Fig. 2C). The slower onset of AICD in response to anti-CD3 plus anti-CD28 paralleled the slower kinetics of p65/RelA loss from the nucleus in response to this stimulus (compare Fig. 1, C and D). In all cases, the period of accentuated cell death coincided with the loss of nuclear p65/RelA for both stimuli. Because c-Rel levels did not decline significantly during this phase, we inferred that c-Rel-dependent antiapoptotic signals could not overcome AICD. Consistent with this idea, c-Rel-deficient splenic T cell blasts underwent AICD as did normal cells (A. Mittal, W. Tam, and R. Sen, unpublished observations). These observations are consistent with the hypothesis that transient nuclear p65/RelA regulates the period of cell viability before AICD. Transient expression of a subset of NF-B target genes A prediction of our hypothesis is that one or more NF-B-regulated antiapoptotic genes must be transiently expressed during T cell activation. Several antiapoptotic genes have been suggested to be NF-B targets, such as IAP family members, Bcl-2 family members, IEX-1, A20, Gadd45, and TNFR-associated factor family members (23, 24). Among these, several have been shown to protect against cell death under specific circumstances. For example, X-IAP protein protects endothelial cells from TNF-␣-mediated cytotoxicity (48), IAP2 has been shown to protect against Jurkat cell death in response to TNF-␣ (49), Bcl-2 family members protect naive B lymphocytes against anti-Ig-induced death (50 – 52), and Gadd45 protects B cells against Fas-induced death (35). To test whether any of these genes were likely candidates to regulate the timing of AICD, we determined their expression profiles as a function of activation time. Total RNA isolated from splenic CD4⫹ T cell blasts activated with PMA and ionomycin for various times was analyzed by quantitative (real-time) RT-PCR assays. The expression of individual genes was normalized to GAPDH RNA at each time point and then compared with that of unstimulated cells. Although all tested genes were putative NF-B targets, and both p65/RelA and c-Rel were induced in these cells, the induction patterns varied considerably (Fig. 3). In particular, only IEX-1, Gadd45, and Bcl-X mRNA followed the pattern of p65 nuclear expression; that is, the mRNA
FIGURE 3. Transient expression of a subset of putative NF-B target genes in activated cells. Splenic CD4⫹ T cell blasts were stimulated with PMA and ionomycin for the indicated times, and total RNA was used for RT, followed by realtime PCR analyses. The genes analyzed are indicated, and the data are represented as the fold induction (y-axis) over the levels present in unactivated cells (0 h). RNA levels of the test genes were first normalized to GAPDH expression and then compared with those in unactivated cells. The results shown represent the mean ⫾ SD from two independent RNA preparations.
of these genes was first up-regulated and then rapidly down-regulated. Importantly, the kinetics of mRNA down-regulation preceded the onset of AICD. These genes were also transiently induced in close correspondence with the timing of nuclear p65 in T cell blasts activated by CD3 and CD28 cross-linking (A. Mittal, unpublished observations). We found no expression of X-IAP in these cells under the conditions examined; IAP2 was expressed in unstimulated cells and was not increased upon activation (A. Mittal and R. Sen, unpublished observations). We next determined the pattern of protein expression of these genes under similar conditions. Of the three genes whose mRNA levels correlated with nuclear p65/RelA, Gadd45 protein levels increased rapidly in response to pharmacological stimulation (Fig. 4A) or anti-CD3 and anti-CD28 stimulation (Fig. 4B), then decreased in parallel with the level of mRNA. Bcl-xL increased slightly in response to PMA and ionomycin and then subsided by 4 h (Fig. 4A), whereas this effect was barely discernible in antiCD3- plus anti-CD28-stimulated cells (Fig. 4B). We also noted elevated levels of a shorter form of Bcl-x, most probably Bcl-xS, at latter stages of activation (Fig. 4, A and B). Recognition of Bcl-xL by this Ab was confirmed in freshly isolated spleen CD4⫹ T cells stimulated with anti-CD3 and anti-CD28 as described by Boise et al. (53) (data not shown). In addition, this Ab detected Bcl-xL in transfected BOSC23 cells (data not shown). Thus, Bcl-x protein expression did not correlate well with the levels of Bcl-x mRNA. IAP2 protein levels, like its mRNA, did not change significantly over the time course of the experiment (Fig. 4). Finally, we were unable to assay IEX-1 expression due to the lack of Abs. Taken together, the mRNA and protein expression data implicate Gadd45 and, perhaps, IEX-1 as possible determinants of the timing of AICD. Extended viability by ectopic expression of p65/RelA-dependent antiapoptotic genes The experiments described above are consistent with the model in which transient p65/RelA expression in the nucleus results in transient expression of a subset of antiapoptotic genes. When these genes are down-regulated, the cells succumb to AICD. The model predicts that extended expression of NF-B-dependent antiapoptotic genes should prolong cell viability. To test this, we expressed Bcl-xL, IEX-1, and Gadd45 in a T cell hybridoma (DC27) and splenic CD4⫹ T cell blasts by retroviral gene transduction. Forty-
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FIGURE 4. Protein expression of putative NF-B target genes in activated cells. Splenic CD4⫹ T cell blasts were stimulated with PMA and ionomycin (A) or CD3 and CD28 Abs (B) for the indicated times, and whole-cell extracts were prepared at the indicated time points. Extracts (60 g) were fractionated by SDS-PAGE and immunoblotted using the indicated Abs. The same blot was probed with the various Abs after stripping. Results are representative of three independent experiments.
eight hours after infection, DC27 cells were activated using PMA and ionomycin, and cell viability was determined at various times thereafter using PI exclusion as described in Materials and Methods. Uninfected cells or control-infected cells underwent maximal apoptosis in the 10- to 18-h period (Fig. 5A). At these times, IEX-1- and Bcl-xL-expressing cells showed somewhat increased viability compared with control cells, whereas Gadd45-expressing cells were significantly more resistant to AICD even compared with IEX-1-expressing cells (Fig. 5A). Because each gene alone conferred some resistance to AICD, we suggest that their coexpression in activated T cells is likely to contribute significantly to cell survival before AICD. These experiments were also conducted in splenic CD4⫹ T cell blasts. Retroviral infection was conducted for 2 days, resulting in ⬃30 –50% infection efficiency on the basis of GFP expression (data not shown). After infection, viable cells were rested for 12–16 h and restimulated with CD3 and CD28 Abs to induce AICD. The proportion of viable GFP⫹ cells was determined by flow cytometry as a function of time. Uninfected or control-infected cells died most rapidly between 8 and 16 h of activation (Fig. 5B). As seen in DC27 cells, Gadd45 greatly enhanced cell viability; however, IEX-1 and Bcl-xL were not significantly protective (Fig. 5B). We evaluated the cell cycle status of control- and Gadd45-infected DC27 and splenic CD4⫹ T cell blasts. In PMAand ionomycin-treated DC27 cells, the proportions of cells in G1, G2, and S phases were very comparable between control- and Gadd45-infected cells (data not shown). Although we observed a small increase in the proportion of G1 phase cells in Gadd45expressing CD4⫹ blasts compared with control-infected cells (data not shown), based on the similarity of anti-apoptotic effects in hybridoma and CD4⫹ blasts, our interpretation is that cell cycle dysregulation is not the major mechanism of Gadd45-dependent cell survival. We propose that of the several possible NF-B-dependent antiapoptotic genes, transient expression of Gadd45 regulates the onset of AICD in activated T cells.
Discussion The observations reported in this study show that p65 and c-Rel behave differently in activated T cells. RelA/p65, which is induced
FIGURE 5. Ectopic expression of IEX1 and Gadd45 reduces AICD. A, DC27 T hybridoma cells were infected with IEX-1- or Gadd45-expressing retroviruses or the respective control viruses (MSCV or MIGR1) or were left uninfected. Infected cells were treated with PMA and ionomycin, and the percent viability of GFP⫹ cells was determined by PI exclusion. The results shown represent the mean ⫾ SD from at least three independent experiments; each experiment was performed in triplicate for every sample and condition. B, Splenic CD4⫹ T cell blasts were infected with the same viruses as in A, rested overnight, and then activated with 1 g/ml plate-bound anti-CD3 and anti-CD28 Abs. At each time point, the viability of GFP⫹ cells was determined using PI exclusion. The percent viability (y-axis) indicates the proportion of GFP⫹PI⫺ cells in the activated cell population compared with unactivated controls, calculated as described in Materials and Methods. These results represent the mean ⫾ SD from at least three independent experiments; each experiment was performed in triplicate for every sample and condition.
in response to TCR signals, but not c-Rel, is effectively removed from the nucleus despite continued stimulation. We attribute the selectivity to two properties of these Rel proteins. First, p65 contains a strong NES that directs it more effectively to the cytoplasm when associated with IB␣. Secondly, new c-Rel synthesis may generate so much protein that the limited pool of newly synthesized IB␣ is unable to effectively down-regulate nuclear c-Rel. It is also interesting to note that TCR plus CD28 signals result in degradation of Bcl-10 (54), an essential signaling intermediate from the TCR to NF-B (55, 56). This mechanism may prevent reactivation of cytoplasmic p65/IB␣ complexes that have been retrieved from the nucleus. Because c-Rel induction in activated T cells circumvents IKK activation, nuclear c-Rel levels continue to increase even after the canonical TCR to NF-B response is terminated due to lack of Bcl-10.
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One of the consequences of p65 removal from the nucleus is that p65-dependent genes are rapidly turned off. The mRNA profile of three putative NF-B-regulated antiapoptotic genes paralleled the kinetics of nuclear p65 expression. Of these, ectopic expression of only Gadd45 provided significant protection against AICD. Despite the kinetics of Bcl-x mRNA expression being consistent with a functional role for the encoded protein, ectopic expression of Bcl-xL did not enhance cell viability. These results parallel previous reports that neither Bcl-2 nor Bfl-1/A1 protects activated T cells from AICD (7, 8, 57), although Bcl-2-like genes protect against activated cell autonomous death (7, 8, 57). The difference can be attributed to the mechanism of Fas-induced death that circumvents the mitochondrial pathway by directly activating caspase 8 (58, 59). Thus, which NF-B-regulated gene provides antiapoptotic function in a particular situation depends on the nature of the death trigger. It is interesting to note that transgenic expression of IEX-1, one of the genes whose expression followed p65 kinetics in our assays, was previously shown to impair apoptosis of activated T cells and increase susceptibility to a lupus-like disease (60, 61). The diminished role of IEX-1 in our in vitro assays may reflect lower expression levels compared with the transgene or accentuation of the effect in the whole animal by other factors. Overall, we infer from these observations that transient expression of a subset of NF-B-dependent antiapoptotic genes may regulate the timing of AICD and thus the duration of the effector phase. NF-B has been previously implicated in providing survival signals to lymphocytes. For example, a role for NF-B is evident in the initial proliferative response of both B and T lymphocytes. B lymphocytes from c-Rel-deficient mice die readily in response to surface Ig cross-linking or gamma irradiation, compared with normal cells. This requirement for c-Rel is based on its activation of Bf1-1/A1 and Bcl-xL genes that provide viability at this stage (50 – 52). NF-B has also been shown to protect naive T cells activated via the TCR. In this situation, NF-B has been proposed to suppress p73 expression, a p53 family member, thereby reducing proapoptotic pathways initiated by p73 (62). Furthermore, apoptosis is also greatly enhanced, and proliferation is significantly impaired, in p50/c-Rel double-deficient T cells in response to TCR stimulation. Much of this loss of viability can be reversed by ectopic expression of Bcl-2 (36). However, it is less clear how the choice between cellular life and death is established. Our observations suggest a mechanism by which the induction and down-regulation of nuclear p65 provide a cell-autonomous timing mechanism that determines the duration of cell viability. Importantly, this mechanism operates in the context of continuous cell stimulation and therefore must be distinguished from mechanisms that lead to apoptosis upon cessation of the stimulus. In the initial phase of the immune response, cells do not undergo AICD despite down-regulation of p65/RelA-dependent genes, probably because the Fas/FasL pathway is inhibited by other factors, such as c-FLIP (14). During IL-2-dependent proliferation, c-FLIP-mediated protection is gradually lost. Upon TCR re-engagement, these cells rely largely upon p65/RelA-dependent antiapoptotic genes, such as IEX-1 and Gadd45, to counteract Fas-induced death. However, NF-B dynamics ensure that the protection is transient, thereby limiting the effector phase of the response and making p65/RelA-dependent survival genes crucial for striking the right balance between continued response or AICD. Recently, a similar situation has been reported in macrophages; Lawrence et al. (63) found that IB-kinase-␣-deficient macrophages maintained nuclear NF-B for a longer time in response to LPS compared with normal cells. As a consequence, several NFB-dependent antiapoptotic genes were expressed for extended periods, and cell death was delayed (63). This resulted in accentuated
cytokine production and morbidity in mice, indicating the importance of the duration of cell activation in vivo. It is possible that an alteration of NF-B kinetics in T cells and consequent dysregulation of AICD may contribute to cell viability in chronic lymphoproliferative diseases.
Acknowledgments We thank Dr. Mei Wu (Boston University School of Medicine, Boston, MA) for the IEX-1 cDNA clone, and Kara Smith for cloning into MSCV. We are grateful to several colleagues, in particular Dr. Joan Press, for helpful comments during the course of this work, and Valerie Martin for preparation of the manuscript.
Disclosures The authors have no financial conflict of interest.
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